Materials Science and Engineering C 30 (2010) 910–916

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Materials Science and Engineering C

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Synthesis of oleic acid-stabilized silver nanoparticles and analysis of theirantibacterial activity

Anh-Tuan Le a,⁎, Le Thi Tam a, Phuong Dinh Tam a, P.T Huy a, Tran Quang Huy b, Nguyen Van Hieu c,A A Kudrinskiy d, Yu A Krutyakov d

a Department of Nanoscience and Nanotechnology, Hanoi Advanced School of Science and Technology (HAST), Hanoi University of Technology, F Building,40 Ta Quang Buu street, Hanoi, Vietnamb National Institute of Hygiene and Epidemiology (NIHE), 01 Yersin, Hai Ba Trung District, Hanoi, Vietnamc International Training Institute of Materials Science (ITIMS), Hanoi University of Technology, No 1 Dai Co Viet, Hanoi, Vietnamd Department of Chemistry, M V Lomonosov Moscow State University, Leninskie Gory, 119991 Moscow, Russian Federation

⁎ Corresponding author. Tel.: + 84 4 3 623 0435; faxE-mail address: tuanla-hast@mail.hut.edu.vn (A.-T. L

0928-4931/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.msec.2010.04.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 August 2009Received in revised form 25 February 2010Accepted 21 April 2010Available online 29 April 2010

Keywords:Silver nanoparticlesColloidal solutionTollens processAntibacterial effectGreen synthesis

The development of new and simple green chemical methods for synthesizing colloidal solutions offunctional nanoparticles is desirable for environment-friendly applications. In the present work, we report afeasible method for synthesizing colloidal solutions of silver nanoparticles (Ag NPs) based on the modifiedTollens technique. The Ag NPs were stabilized by using oleic acid as a surfactant and were produced for thefirst time by the reduction of silver ammonium complex [Ag(NH3)2]+(aq) by glucose with UV irradiationtreatment. A stable and nearly monodisperse aqueous Ag NPs solution with average-sized particles (~ 9–10 nm) was obtained. The Ag NPs exhibited high antibacterial activity against both Gram-negativeEscherichia Coli (E. coli) and Gram-positive Staphylococcus aureus bacteria. Electron microscopic images andanalyses provided further insights into the interaction and bactericidal mechanism of the Ag NPs. Theproposed method of synthesis is an effective way to produce highly bactericidal colloidal solutions formedical, microbiological, and industrial applications.

: + 84 4 3 623 0293.e).

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The development of functional nanoparticles (NPs) has beenintensively pursued for a variety of technological applications [1].There are two different approaches to synthesize NPs: the “top-down”approach, which utilizes physical methods, and the “bottom-up”approach, which employs solution-phase colloidal chemical methods.The physical methods have the advantage of being able to producelarge quantities of NPs but have the disadvantage of an inability tocontrol completely the distribution of particle sizes. Colloidalchemical synthesis methods, on the other hand, have shown to becapable of synthesizing uniform NPs with desired particle sizes [2].For this reason, the latter methods are more widely used in preparinga robust variety of nanocrystals [3].

It has been reported recently that silver in the form of nanoparticles(Ag NPs), especially for medical applications, is a promising alternativeto silver salts and bulkmetal. Saltsmay release the silver too quickly anduncontrollably, while bulk metal is too inefficient a releasing material.Ag NPs exhibit outstanding physical properties that are obviouslydifferent from those of ions and bulk metal, showing promise of their

use in many applications in the fields of medicine, microbiology, andanalytical chemistry, among others [4]. More importantly, Ag NPs arehighly antimicrobial due to their antiseptic properties against severalspecies of bacteria, including the common kitchenmicrobe. As such, AgNPs have caught the attention of many researchers, especially becauseof their extraordinary antimicrobial activity [5–7].

A synthesis method using the Tollens process has been proposedfor synthesizing Ag NPswith controlled size [8,9]. The basic reaction inthis process involves the reduction of a silver ammoniacal solution byusing either aldehydes or reducing sugars. A fundamental reductionreaction involving the Tollens process is as follows:

Ag NH3ð Þ2� �þ aqð Þ + RCHO aqð Þ→Ag sð Þ + RCOOH aqð Þ:

Various reduction reagents, such as formaldehyde, glucose,galactose, maltose, and lactose, are used to prepare these Ag NPs[10]. The size of Ag NPs strongly depends on the ammoniaconcentration and the pH of the medium during the reductionprocess [10]. Under optimal conditions, Ag NPs with an average size of25 nm can be obtained using maltose as a reducing reagent and SDS,Tween 80, or the polymer PVP 360 as the stabilizer [11]. Theantibacterial action of Ag NPs is strongly dependent on their size,dosage, and morphology. For such applications, Ag NPs should besufficiently small to be capable of penetrating through cell

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membranes to affect intracellular processes fromwithin the cell. Thus,it is essential to develop suitable synthesis methods to improve theantibacterial activity of Ag NPs through the modification of their size.To this end, new stabilizing agents have been found to bring about thereasonable improvement of the stable dispersion of Ag NPs.

The overall aim of the present work is to develop a new chemicalapproach for producing more stable aqueous dispersions of Ag NPsusing the modified Tollens technique. In comparison with other Tollensmethods involving the use of toxic solvents and highly reactivechemical-reducing agents, this newly developed technique is simple,cost-effective, and environmentally safe because common and simplechemicals are used. In particular, Ag NPs stabilized with oleic acid areproduced for the first time and their antibacterial activities areinvestigated. Oleic acid-stabilized Ag NPs show high antibacterialactivity against both theGram-positive humanpathogen Staphylococcusaureus (S. aureus) and Gram-negative bacteria Escherichia coli (E. coli).Electron microscopic studies reveal insights into the interactionmechanism between the Ag NPs and tested bacteria in question.

2. Experimental procedures

2.1. Synthesis method

All reagents were of analytical grade and used without furtherpurification. In a typical experiment, 1.7 g (1.0×10−2mol) of silvernitrate (AgNO3) (Aldrich, 99.9+%) was dissolved in 100 mL ofdeionized water. The AgNO3 solution was then precipitated with0.62 g (1.55×10−2mol) of sodium hydroxide (Aldrich, 99+%).

The obtained precipitate, composed of Ag2O, was filtered anddissolved in 100 mL of aqueous ammonia (NH3) (0.4% w/w,2.3×10−2mol) until a transparent solution of silver ammoniumcomplex, [Ag(NH3)2]+(aq), formed. Next, 2.5 g (8.9×10−3mol) ofoleic acid (Sigma-Aldrich, 99+%) was added dropwise into thecomplex, and the resulting solution was gently stirred for 2 h atroom temperature until the complete homogeneity of the reactionmixture was achieved. Finally, 2 g (1.11×10−2mol) of glucose wasadded to the mixture at room temperature with gentle stirring.The reduction process of the silver complex solution (in a quartzglass) was initiated with UV irradiation. UV treatment was carriedout for 8 h under vigorous stirring without additional heating. AUV lamp (λ=365 nm, 35 W) was used as a light source tostimulate the reduction process. After 8 h of irradiation, atransparent dispersion of oleic acid-stabilized Ag NPs wasobtained. The synthesis of the Ag NPs was successfully conductedwith a final Ag concentration in the range of 0.1–2%.

2.2. Characterizations

The UV–vis absorbance spectra of the Ag NPs were recorded using anHP 8453 spectrophotometer. Quartz cuvettes with a 10 mm path lengthwere used for the measurement of dispersion spectra. A characteristicfeature of absorption spectra of nano-sized silver particles was deter-mined by the formation of an intense and broad band in the visible rangein which this band is called the surface plasmon resonance (SPR). X-raydiffraction was carried out on a Dron-3 machine using CuKα radiation(λ=0.154 nm) at a step of 0.02º (2θ) at room temperature. Thebackground was subtracted with the linear interpolation method.

The electronic images and diffractograms of the AgNPswere recordedon JEM1010 (JEOL, Japan) and Leo 912ABOmega (Leo Ltd.) transmissionelectron microscopes (TEM) operating at 80 kV. The samples for TEMcharacterizationwere prepared by placing a drop of the colloidal solutiononto a formvar-coated copper grid, which was then dried at roomtemperature. All size distributions were calculated using the FemtoscanOnline v. 2.2.91 software (Advanced Technologies Center, Russia).

2.3. Antibacterial analysis

The antibacterial activity of Ag NPs was tested against Gram-negativeE. coli and Gram-positive S. aureus bacteria. These bacteria were culturedon a Luria-Bertani (LB) liquid nutrient broth medium with pH=7. Theculture mediumwas incubated at 37 °C for 24 h, the bacterial concentra-tion would reached 108 colony-forming units (CFUmL−1).

The standard dilution micromethod was used for performing theantibacterial activity tests on agar plates. Aqueous dispersions of Ag NPsof varying concentrations (from 0 to 10 μg mL−1) were prepared fromthe initial silver colloidal solution. To obtain a uniform distribution, thenutrient agar medium was heated to 50 °C. Next, 10 mL of eachnanosilver solution was added onto Petri plates containing 25 mL ofnutrient agar medium. The total volume on each Petri plate was kept to35 mL, and the mixture was solidified with agar after 15 min. A 100μLsuspensionof E. coli bacteriawaspipetted and spreadonto the surfaceofthe agarmedium containing silver NPs. The Petri plates were incubatedat 37 °C for 24 h in a shaking incubator (150 rpm) to encourage bacterialcell growth. The intensity of the bacterial growth on the agar plateswithsilver NPs of variable concentration was monitored by the naked eyeand stereo microscope (ZMS800, Nikon). All the obtained results werecomparedwith the bacteria growth intensity on an agar plate on whichno Ag NPs were applied.

The viable bacteria were monitored by counting the number of thecolony-forming units from the appropriate dilution of bacteriaconcentration on the agar plates. A control sample test was alsoconducted for comparison. The control sample included a reactionmixture after UV treatment with no addition of AgNO3 and containedNH3, oleic acid, and glucose. All experiments were performed understerile conditions and in triplicate.

3. Results and discussion

3.1. Formation of Ag NPs

To determine the optimum conditions for the synthesis of thesurfactant-stabilized Ag NPs, several experiments were carried out inwhich the concentrations of the reagents and the order of their additionto the reaction mixture were varied. A two-step reduction process ofsilver ammonium complex (Ag+) to silver metal (Ag0) was performed:

In the first step, the dropwise addition of the oleic acid as astabilizer was performed to obtain a stable sol with high homogeneityand viscosity. The second stepwas performed to reduce the oleic acid-stabilized silver complex to silver metal in the presence of UVirradiation treatment. UV irradiation causes the excitation of [Ag(NH3)2]+ ions followed by electron transfer from the glucosemolecule to Ag+, thus producing Ag0 atoms that then form clustersand seeds. We propose a possible mechanism for the formation andgrowth of Ag NPs as follows. Primarily, UV treatment leads to thesubstantially simultaneous formation of a large amount of silvernuclei. These nuclei are formed with a homogeneous distributionthroughout the solution and tend to aggregate to form biggerparticles. The remaining silver ions are adsorbed onto the surface ofthe already-formed particles, thus bringing about successive

Fig. 1. (a) As-synthesized Ag NPs colloidal solutions at different concentrations. (b) TheUV–visible absorption spectrum of a typical colloid sample. The maximum absorbancepeak is observed at 420 nm.

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reduction and attracting oppositely charged surfactant ions. Thesesurfactant ions form a capping layer on the surface of the silverparticles. The surfactant molecules inhibit this aggregation associationthrough the capping/template effect and thus acts as a particlestabilizer [12,13]. It should be noted that the hydroxymethylfunctionalities of the surfactant molecules anchor the molecule tothe cluster surface, while the hydrophobic chain protects the clusterfrom aggregation with its next neighbor due to electrostatic repulsionand steric hindrance, thus inhibiting coalescence [13].

As an example, the as-synthesized samples of Ag NP colloidalsolutions with different silver concentrations are presented in Fig. 1(a). With increasing silver concentration, the color of the solutionchanged from light yellow to dark yellow to brown. It should be notedthat the change in color reveals the formation of Ag NPs in the solution[8]. The existence of Ag NPs in the solution was also verified by TEMimages. The Ag NP colloids were found to be very stable and nosedimentation formed even after storing for several months. Asobserved in Fig. 1(b), an optical absorption band with a maximum at420 nm was found. This is a typical feature of the absorption ofmetallic Ag NPs due to the SPR, indicating the presence of Ag NPs inthe solution. The appearance of an SPR band is the result of theinteraction between the incident light on the nanoparticle surface and

Fig. 2. TEM images of Ag NPs colloids with two fatty acids as the stabilizer agents: (a)myristic acid and (b) oleic acid. (c) The XRD patterns of Ag NPs powder stabilized withmyristic and oleic acids.

Table 1Comparison of the characteristic properties of Ag NPs prepared with myristic and oleicacids.

Characterizations Ag NPs stabilized withmyristic acid

Ag NPs stabilized witholeic acid

Average particle size (nm) 6–7 9–10Size distribution Polydispersity Uniform dispersionMaximum absorbancewavelength (nm)

415 420

pH 12–14 9–14*MIC (mcg/ml) 8 2

* Minimum inhibitory silver concentration (MIC) leading to inhibition of bacterialgrowth.

Fig. 3. Tested bacteria grown on agar plates with different concentrations of Ag NPs. Thetwo top lines [(i) and (ii)] correspond to the Gram-positive S. aureus bacteria, while thetwo bottom lines [(iii) and (iv)] correspond to the Gram-negative E. coli bacteria. Notethat lines (i) and (iii) are samples with low Ag NPs concentrations, whereas lines(ii) and (iv) are samples with high Ag NPs concentrations.

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the electrons of the metal [6]. As such, changes in the electron densitynear the silver surface result in changes in the SPR band position.

3.2. Structure of Ag NPs

To select an effective stabilizer, two fatty acids, myristic acid andoleic acid, were employed as the stabilized agents. The TEM and XRDanalyses of the structure and particle sizes of the stabilized Ag NPswere displayed in Fig. 2(a)–(c). It was found that myristic acid-stabilized Ag NPs possessed small particles (~6–7 nm) and amaximum optical absorbance peak at 415 nm. It can be also seenfrom Fig. 2(a) that the myristic acid-stabilized Ag NPs exhibited highpolydispersion. Moreover, it should be noted that the colloids of themyristic acid-stabilized Ag NPs were only stable at high pH conditions.This restricts the applicability of these colloids in the industry. Incontrast, oleic acid-stabilized Ag NPs had larger particle sizes ~9–10 nm [Fig. 2(b)], and were very stable in a wide range of pHconditions (see Table 1). Thus, this type of Ag NPs colloids showpromise for potential solution-phase applications.

The crystallinity and changes in sizes of the silver particles wasverified by the powder X-ray diffraction as shown in Fig. 2(c). Itshowed narrow peaks assigned to 111, 200, and 220 planes of a facecentered cubic lattice of bulk silver. This fact confirmed that thesynthesized NPs consisted of pure silver with high crystallinity. Ourcalculations from the XRD patterns according to Scherrer expressionrevealed that, the oleic acid-stabilized Ag NPs had larger average-sized particles (~10–11 nm) compared to the myristic acid-stabilizedAg NPs (~7–8 nm). This result is fully consistent with the calculationfrom TEM images. Furthermore, the broadening of the full width athalf maximum (FWHM) of the XRD pattern revealed the decrease inthe sizes of silver particles. In comparison with the previously usedTollens processes, Ag NPs stabilized with oleic acid formed smalleraverage particle diameters (~9–10 nm) compared with Ag NPsstabilized with SDS, Tween 80, or polymer PVP 360 (~25 nm) [11].Oleic acid is also inexpensive, easy to use, and harmless toenvironment materials [14]. As observed in Fig. 2(b), the oleic acid-stabilized Ag NPs are well-formed and nearly uniformly dispersed,while most of the Ag NPs are discrete with moderate degrees ofpolydispersion. This confirms the nanocrystalline character of theparticles and the low degree of their polydispersity. More importantly,almost no aggregates of Ag NPs were found.

To determine the stability of Ag NPs in aqueous media, theabsorbance intensity of the Ag NP colloidal solution was monitoredover time. UV–vis spectra and TEMmeasurements were conducted toidentify the changes in particle size after the aging of Ag NPs. Resultsrevealed that the particle size remained almost unchanged after beingstored for two months (data not shown here). This finding suggeststhat as-prepared aqueous dispersions of Ag NPs are very stable againstaggregation for several months. These results also imply thatreproducible dispersions of silver colloids can be created in batchesand stored. Moreover, the addition of appropriate amounts of oleicacid as a stabilizing agent to silver colloids not only improves theirstability but also allows duplicate measurements to be made with lessdeviations.

3.3. Antibacterial activity of Ag NPs and its mechanism

Both myristic and oleic acid-stabilized Ag NPs were subjected toantimicrobial tests. The Ag NP solutions were added to the nutritionmedia to obtain 10–100 times dilutions of the Ag NPs in the media.

Fig. 3 displays the images of the agar plates taken for the antibacterialactivity against S. aureus bacteria [see two top lines: (i) and (ii)] and E. colibacteria [see two bottom lines: (iii) and (iv)]. It was found that bacteriagrewdrastically after 24 h for the control samples [see Fig 4(a) and (c)]. Asthesilveramount increased, theactivityof suppressionofbacterial growthincreased. The bacterial cell colonies on the agar-plates were detected by

viable cell counts, which were the counted number of colonies thatdeveloped after a sample was diluted and spread over the surface of anutrient medium solidified with agar and contained in a petri dish. Thenumber of colony-forming units (CFU) reduced significantly with theincrease in silver concentration. When the silver content reached theoptimal concentration, complete inhibition in the bacteria growth wasobserved.

The reproducibility of antibacterial activity was investigated byperforming the antibacterial tests in triplicate, and the average value ofconcentration was assigned a relative standard deviation of 5%. No CFUwas observed in the oleic acid-stabilized Ag NPs sample loading of2.0 μg mL−1 and higher [see Fig. 4(b) and (d)]. This reveals that theoptimal condition for the complete inhibition in the bacterial growthwasof aminimumsilver concentration at 2.0 μg mL−1for both the E. coli and S.aureus bacteria. The antibacterial activity of the oleic acid-stabilized AgNPs was higher up to 4 times against the E. coli and S. aureus bacteriacompared with themyristic acid-stabilized Ag NPs (Table 1). The presentwork highlights that the silver concentration for the effective suppressionin thebacterial growthwas found to bemuch lower than that described inprevious reports ~5–20 μg mL−1 [15,16]. Thus, this can lead toa significantreduction in the used silver content and consequently to the reduction ofthe total prices of the final products.

Apart from these findings, the tested colloidal solutions of Ag NPshad bactericidal effects, which cause not only the inhibition ofbacterial growth but also of the bacteria to be killed. The variousconcentrations required for growth inhibition or for the killing of thebacteria were determined by the biological properties of individualbacterial species. The structural difference lies in the organization ofpeptidoglycan (PG), which is the key component of the membranestructure. It should be noted that Gram-positive bacteria are able toretain the crystal violet stain because of the high amount of PG in the

Fig. 4. Agar plates samples treated (a) without Ag NPs and (b) with Ag NPs correspond to Gram-negative E. coli bacteria; (c) without Ag NPs and (d) with Ag NPs correspond to Gram-positive S. aureus bacteria. In the presence of Ag NPs, bacterial growth was completely inhibited, whereas the control samples, those that were not added with Ag NPs, showed adrastic growth of bacteria.

Fig. 5. TEM images of E. coli showing the interaction stages of Ag NPs with the bacteria after (a) 0 min, (b) 30 min, (c) 1h, and (d) 2h.

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cell wall. Gram-positive cell walls typically lack outer membranes.Their structure mainly comprises of a cytoplasmic lipid membraneand a thick PG layer. However, the Gram-negative bacteria onlyexhibit a thin layer of peptidoglycan (about 2–3 nm) between thecytoplasmic membrane and the outer cell wall. The outer membraneof the Gram-negative cells is predominantly constructed from tightlypacked lipopolysaccharide (LPS) molecules, which provide aneffective permeability barrier. In the present work, it was shownthat the antibacterial activity of Ag NPs with the Gram-negativebacterium E. coli exhibited a quicker response than that with theGram-positive bacterium S. aureus. This could be understood by thesignificant difference in thickness of the PG layer of the bacteriastrains [see Figs 5(a) and 6(a)].

To gain more rudimentary insights into the interaction andbactericidal mechanism between the Ag NPs and the E. coli and S.aureus bacteria, the electron microscopic technique was conducted.The colloidal solution was dropped onto the surface of E. coli and S.aureus grown on agar plates. After 30 min, 1 h, and 2 h, E. coli and S.aureuswere taken out and underwent the sectioning method for TEMobservation. At differentmagnifications and sections [Figs. 5–6], manyAg NPs bindings around both the E. coli and S. aureus cell membranesas well as inside the cells were found. As observed, the Ag NPs firstattached to the surface of the cell membrane, penetrating furtherinside the bacteria. It should be noted that only Ag NPs withsufficiently small diameters penetrated into the cells. The cytoplasmwas destroyed as the Ag NPs penetrated the cell [Figs 5(c) and 6(c)].This proves how E. coli and S. aureus cells can be eliminated by the AgNPs. The changes took place in the cells’ membrane morphology,producing a significant increase in their permeability. This affects theproper transport through the plasma membrane, leaving the bacterialcells incapable of properly regulating transport through the plasmamembrane, resulting eventually to cell death [17]. As shown in Figs. 5(b,d) and 6 (b,d), in addition to being fixed to the cell membrane, theAg NPs are capable of penetrating through it to be distributed inside abacterium. After interacting with the E. coli and S. aureus bacteria, theAg NPs adhered to the cell wall of the bacteria and penetrated the cellmembrane, resulting in the inhibition of bacterial cell growth andmultiplication.

Fig. 6. TEM images of S. aureus showing the interaction stages of Ag NP

It should be noted that the Ag NPs exhibited a high antibacterialeffect due to their well-developed surface, which provides maximumcontact with the environment. Moreover, the as-prepared Ag NPswere sufficiently small and capable of penetrating though the cellmembrane to affect the intracellular processes from the inside.Therefore, it is reasonable to claim that the binding of the particlesto the bacteria depends on the surface area available for theinteraction. Smaller particles with the larger specific surface areaavailable for the interaction have more bactericidal effect than thelarger particles [18].

More noticeably, the increase in bacterial resistance to antimicro-bial agents poses a serious problem in the treatment of infectiousdiseases as well as in epidemiological practice. Increasingly newbacterial strains have emerged with dangerous levels of resistance,including Gram-positive and Gram-negative bacteria. Dealing withbacterial resistance requires precautions that can lead to theprevention of the emergence and spread of multiresistant bacterialstrains and the development of new antimicrobial substances. Theresults of this study clearly demonstrate that oleic acid-stabilized AgNPs can effectively inhibit the growth and multiplication of the testedbacteria. This highly active antibacterial activity was observed at verylow Ag concentrations of about 2.0 μg mL−1. The excellent antibac-terial activity against the E. coli and S. aureus bacteria even at a lowsilver loading makes Ag NPs very ideal for a highly cost-effective anti-microbial solution with long-lasting effect in green industrialapplications.

4. Conclusions

Ag NPs stabilized with oleic acid were successfully synthesized forthe first time by using the modified Tollens technique. It was shownthat the colloids of synthesized Ag NPs could exist in the form of verystable aqueous dispersions for several months without aggregates orsedimentation. It was also presented that oleic acid-stabilized Ag NPshad high levels of antibacterial activity against the Gram-positivehuman pathogen S. aureus and Gram-negative bacteria E. coli. Electronmicroscopic techniques helped elucidate the interaction mechanismbetween the Ag NPs and the bacteria in question.

s with the bacteria after (a) 0 min, (b) 30 min, (c) 1h, and (d) 2h.

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Acknowledgments

This workwas financially supported by the research project (GrantB2008-01-155) funded by Vietnamese Ministry of Education andTraining and application-oriented research basic project (Grant 05/09-ĐTĐL). A.T. Le wishes to acknowledge Prof. Phung Dac Cam of theNational Institute of Hygiene and Epidemiology for his assistance withthe antibacterial analysis techniques. We would also like to thank Dr.Manh-Huong Phan of the Department of Physics in the University ofSouth Florida, USA, for editing this manuscript.

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